A retroviral expression system based on tetracycline-regulated tricistronic transactivator/repressor vectors for functional analyses of antiproliferative and toxic genes

Abstract

Establishment of stably transfected mammalian cells with conditional expression of antiproliferative or proapoptotic proteins is often hampered by varying expression within bulk-selected cells and high background in the absence of the inducing drug. To overcome such limitations, we designed a gene expression system that transcribes the tetracycline-dependent rtTA2-M2-activator, TRSID-silencer, and selection marker as a tricistronic mRNA from a single retroviral vector. More than 92% of bulk-selected cells expressed enhanced green fluorescent protein or luciferase over more than three orders of magnitude in an almost linear, dose-dependent manner. To functionally test this system, we studied how dose-dependent expression of p27Kip1 affects proliferation and viability of SH-EP neuroblastoma cells. Low to moderate P27Kip1 expression caused transient G0-G1 accumulation without reduced viability, whereas high p27Kip1 levels induced significant apoptosis after 72 hours. This proves that this expression system allows concentration-dependent analysis of gene function and implicates p27Kip1 as a critical regulator of both proliferation and apoptosis in SH-EP neuroblastoma Cells. Copyright © 2006 American Association for Cancer Research.